Microorganisms for treatment or prevention of corpulence and diabetes mellitus, and pharmaceutical composition containing the same

ABSTRACT

The present invention relates to microorganisms for the treatment or the prevention of obesity or diabetes mellitus, which reduce the amount of monosaccharide or disaccharide which may be absorbed into human body by converting monosaccharides such as glucose, fructose, galactose et al. and disaccharides into polymeric materials which cannot be absorbed by the intestine, and relates to a pharmaceutical composition containing the said microorganisms.

FIELD OF THE INVENTION

[0001] The present invention relates to microorganisms for preventing ortreating obesity or diabetes mellitus, which are capable of reducing anamount of monosaccharides or disaccharides that can be absorbed into theintestine by converting those mono or disaccharides into polymericmaterials that cannot be absorbed in the intestines. The presentinvention also relates to use of the microorganisms for preventing ortreating obesity or diabetes mellitus and a pharmaceutical compositioncontaining the microorganisms.

BACKGROUND OF THE INVENTION

[0002] Obesity is well known as a chronic disease caused by variousfactors whose origins have not yet been clearly discovered. It isunderstood that obesity induces hypertension, diabetes mellitus,coronary heart disease, gall bladder disease, osteoarthritis, sleepapnea, respiratory disorder, endomerial, prostate, breast and coloncancer and the like.

[0003] According to the NIH Report (THE EVIDENCE REPORT: ClinicalGuideline on the Identification, Evaluation, and Treatment of Overweightand Obesity in Adults, 1999, NIH), about 97,000,000 Americans sufferfrom overweighting and obesesity, and the number of patients of type IIdiabetes mellitus associated with obesity, reaches about 15,700,000.Moreover, it is reported that about 200,000 people die of diseasesassociated with obesity each year (Dan Ferber, Science, 283,pp 1424,1999).

[0004] Diabetes mellitus is one of the most widespread chronic diseasesin the world, which impose a substantial expense on the public as wellas on patients of diabetes mellitus and their families.

[0005] There are several types of diabetes mellitus that are caused byvarious etiological factors and whose pathogenesis is different fromeach other. For example, genuine diabetes mellitus is characterized byhigh level of blood glucose and glycosuria, and is a chronic disorder ofcarbohydrate metabolism due to a disturbance of the normal insulinemechanism.

[0006] Non-Insulin-Dependent Genuine Diabetes Mellitus (NIDDM), or thetype II diabetes mellitus is found in adults who have insulin-resistancein a peripheral target tissue, despite of normal generation and functionof insulin. Non-Insulin-Dependent Genuine Diabetes Mellitus(NIDDM) canbe caused by three important metabolic disorders, i.e.,insulin-resistance, fucntional disorder of insulin secretion stimulatedby nutrients, and overproduction of glucose in liver. Failure to treatNIDDM, resulting in losing control of blood glucose levels, leads todeath of patients from diseases such as atherosclerosis, and/or maycause late complications of diabetes, such as retinopathy, nephropathyor neuropathy.

[0007] Accompanying diet-exercise therapy, NIDDM therapy usessulfonylurea and biguanidine compounds to control blood glucose levels.Recently, therapeutic compounds such as metformin or acarbose have beenused for treating NIDDM. However, diet-exercise therapy alone or evencombined with chemotherapy using such compounds fails to controlhyperglycemia in some of the diabetes mellitus patients. In such cases,these patients require exogenous insulin.

[0008] Administration of insulin is very expensive and painful topatients, and furthermore, may cause various detrimental results andvarious complications in patients. For example, incidences, such as,miscalculating insulin dosage, going without a meal or irregularexercise, may cause insulin response (hypoglycemia) and sometimes theinsulin response occurs even without any particular reasons. Insulininjection may also cause an allergy or immunological resistance toinsulin.

[0009] There are several methods for preventing or treating obesity ordiabetes mellitus, including diet-exercise therapy, surgical operationand chemotherapy. Diet-exercise therapy involves a low-calorie andlow-fat diet accompanying aerobic exercise, but this therapy requiring aregular performance is hard to continue until achieving the goal.

[0010] Despite of instant effects, a surgery for physically removingbody fat has limitations due to the risk and cost involved in a surgicaloperation and insufficient durability of the effects.

[0011] As one of the most promising therapies currently developed,pharmacotherapy can reduce blood glucose level, inhibit absorption ofglucose, strengthen the action of insulin or induce the decrease ofappetite. The medicines that have been developed so far use variousphysiological mechanisms for the prevention and the treatment of obesityand diabetes mellitus.

[0012] Some medicines, such as, sulfonylurea, metformin, pioglitazone orthiazolidindione derivatives and the like have been developed to enhancethe function of insulin. Although sulfonylurea stimulatesinsulin-secretion from β-cells in the pancreas, it may accompany sideeffects, such as hypoglycemia resulting from lowering blood glucoselevels under normal levels.

[0013] Metformin is mainly used for insulin-nondependent diabetesmellitus patients who fail to recover after diet-exercise therapy. Thismedicine inhibits hepatic gluconeogenesis and enhances glucose disposalin muscle and adipose tissue. However, it suffers from side effects,such as, nausea, vomiting and diarrhea.

[0014] Pioglitazone developed by Takeda in Japan, enhances the functionof insulin through increasing susceptibility of cells to insulin(Kobayashi M. et al., Diabetes, 41(4), pp 476-483, 1992).

[0015] Beta 3-adreno receptor inhibitor (BRL-35135) known as a medicinethat stimulates the decomposition of body fats and that convert bodyfats into heat with a specific action on adipose cells, also suffersfrom lowerings blood glucose level.

[0016] The inhibitor of a pancreatic lipase (Orlistat produced by Rocheof Switzlend) inhibits and/or reduces absorption of body fats byinhibiting pancreatic lipase. It, however, accompanies undersirableinhibition of absorption of fat-soluble vitamin and may also causebreast cancer.

[0017] Generally, medicines that decrease appetite affects catecholaminein the brain. However, dexfenfluororamine and fenfluoroamine have sideeffects of nerve toxicity and valvular heart disease. Also, sibutraminehas side effects of increasing heart rate and blood pressure.

[0018] α-Glucosidase inhibitor (Acarbose produced by Bayer of Germany),is known as a glucose absorbing inhibitor. Acarbose ispseudo-monosaccharide which competitively inhibits the action of variousα-glucosidases existing in microvilli of the gastrointestinal tract.However, taking a large amount of these may induce diarrhea. (W. Puls etal., Front. Horm. Res. 2, 235, 1998).

[0019] Amylase inhibitor that inhibit converting carbohydrates intooligosaccharides has been developed to prevent imbalance of metabolismoriginated from excessive uptake of nutrient. (Sanches-Monge R. et al.Eur. J. Biochem., 183, 0037-40, 1989).

[0020] Dietary fiber using diet with a large amount of vegetable fiberis the easiest way to obtain inhibitory effect on obesity by loweringglucose and/or fat amounts absorbed in the intestine. However, suchmethod also involves problems in requiring facility and manpower for theproduction of dietary fiber with low productivity.

[0021] Polymeric materials, such as, isomaltotriose, dextran andpullulan, inhibit the increase of blood glucose level originated fromglucose. However, such materials also cause severe side effects. Forexample, dextran may induce excessive bleeding by delaying a bloodcoagulation time.

[0022] Among said various medicines, dietary fibers are the most usefulmedicine for prevention or treatment of obesity because no damage to thehuman metabolism-balance and use natural substances.

[0023] Microorganism dietary fiber is produced using microorganisms,such as, Gluconobacter sp., Agrobacterium sp., Acetobacter xylinum, A.hansenii, A. pasteurianus, A. aceti, Rhizobium sp., Alcaligenes sp.,Sarcina sp., Streptococcus thermophilus, Lactococcus cremoris,Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus sake,Lactobacillus reuteri, Lactobacillus lactis, Lactobacillus delbrueckiisubsp., Lactobacillus helveticusglucose var. jugurti, Leuconostocdextranicum, Bulgariscus sp., Campestris sp., Sphingomonas sp.

[0024] Dietary fiber produced by these microorganisms is used asstabilizer, thickening agent, emulsifier, hygroscopic agent of variousfoods and raw materials of cosmetics and pharmaceuticals. Microorganismcellulose, xanthan, acetan, guar gum, locust bean gum, carrageenan,alginate, and agar obtained from seaweed are commercialized.

[0025] Lactobacillus sp. strain is the major component of normalmicrobial flora in the human intestines. Its significant roles formaintaining digestive organ and for healthy environment of the vagina,have been well known. [Bible, D. J., ASM News, 54:661-665, 1988; Reid G.and A. W. Bruce, In H Lappin-Scott (de.), Bacterial biofilms, CambridgeUniversity Press, Cambridge, England, p. 274-281, 1995; Reid G., A. W.Bruce, J. A. McGroarty, K. J. Cheng, and J. W. Costerton, clin.Microbiol. Rev., 3:335-344, 1990]. Generally, Lactobacillus straininhabits in digestive organs (L. acidophilus, L. intestinalis, L.johnsonii, L. reuteri et al.,), muscosa of the vagina (L. vanginals, L.gasseri), food (wine-L. hilgardii), lactobacillus beverage (L. kefir, L.kefiranofaciens), cheese (L. casei), vinegar (L. acetotolerance), theoral cavity (L. oris), yeast (L. sake, L. homohiochi), fruit juice (L.kunkeei, L. mali, L. suebicus), fermented sausages or fish (L.farciminis, L. alimentarious) et al.,

[0026] Many people take health complementary food containing aLactobacillus sp. strain in order to maintain healthy intestines and toprevent urogenital tract infection. Recently, in addition to theprevention of the diarrhea, constipation and urogenital tract infection,various probiotic activities of Lactobacillus, such as, control ofimmunity, control of cholesterol level in blood, prevention of cancer,treatment of rheumatism, alleviation of sensitivity on lactose or effectfor atopic dermatitis, have been reported and thus, have attracted moreattention.

[0027] According to the U.S. Public Health Service Guideline, all of the262 Lactobacillus deposited in ATCC are classified as “Bio-safety Level1,” which stands for no potential risk, which has been known up to now,causing diseases in human or animals. There is no harm to human bodyamong approximately 60 strains of Lactobacillus.

[0028] Recently, there has been a rapid progress in the research for anextracellular dietary fiber produced by Lactobacillus. It has beenreported that a process of producing dietary fiber in these strains arevery complicated because a lot of genes are mediated in the process, andthe amount of dietary fiber thus produced are very low (Int. J. FoodMicrobiol., March 3 40:1-2, 87-92, 1998; Current Opinion inMicrobiology, 2:598-603, 1999; Appl. Environ. Microbiol., February 64:2,659-64, 1998; FEMS Microbiol. Rev. April 23:2 153-77, 1999; FEMSMicrobiol. Rev. September 7:1-2, 113-30, 1990).

[0029] Also, various researches on the synthesis of cellulose byAcetobacter sp. which is well known as a microorganism producing dietaryfiber, have been performed (Aloni Y., cohen R., Benziman M., Delmer D, JBiological chemistry, 171:6649-6655, 1989; Ascher M., J. Bacteriology,33:249-252, 1937; Benziman M., Burger-Rachamimv H., J., Bacteriology,84:625-630, 1962; Brown A M. Journal of Polymer science, 59:155-169,1962; Brown A M, Gascoigne J A, Nature, 187:1010-1012, 1960; Calvin J R,Planta D P, Benziman M., Padan E, PANS USA, 79:5282-5286, 1982; Dehmer DP. Brown R M Jr., Cooper J B, Lin F C, Science, 230:82-825, 1985).

[0030] Acetobacter is a strict aerobe but has characteristics ofsurviving and living under the condition of infinitesimal oxygen, and ofbeing floated to seek for oxygen by means of synthesizing cellulosedietary fiber itself under this condition of infinitesimal oxygen.According to the research regarding the amount and rate of convertingglucose into cellulose dietary fiber by Acetobacter (Brown et al.: Proc.Natl. Acad. Sci. USA, Vol 73 (12), 4565-4569), Acetobacter convertsglucose into cellulose with the speed rate of 400 mol/cell/hour. This isequivalent to the rate that about 200 g glucose can be converted intocellulose dietary fiber by 4×10¹⁵ cells per an hour.

[0031] Although Acetobacter that can metabolizes saccharose is rare,Acetobacter converting sacchores in glucose, exists in nature (PNAS, 9:pp 14-18). Presently, FDA of the United States has approved Acetobacterxylinum for synthesizing acetic acid and sorbose, and has classified itas generally safe microorganism (GRAS: Generally Recognized As Safe).

[0032] As mentioned above, although there have been various researchesand efforts to develop drugs for treatment or prevention of obesity anddiabetes mellitus, their results were not satisfactory. Various chemicalsubstances mentioned above, have been developed for treatment of obesityand diabetes mellitus, but still suffer from various side effects. Thesedrugs forcibly discharge body fat together with valuable proteins.Consequently, any one single drug for treatment or prevention of obesityand diabetes mellitus at the origin thereof does not exist yet.

SUMMARY OF THE INVENTION

[0033] Therefore, the object of the present invention is to providemicroorganisms capable of living within the intestines and convertingoligosaccharides produced by the digestive enzymes into non-digestablepolysaccharides, and thereby remarkably reducing the amount ofoligosaccharide absorbed into the intestines.

[0034] Another object of the present invention is to provide apharmaceutical composition comprising at least one of saidmicroorganisms in an amount effective to prevent or treat obesity anddiabetes mellitus and a pharmaceutically acceptable carrier. Anotherobject of the present invention is to provide a method for preventing ortreating obesity, diabetes mellitus comprising administering to asubject in need thereof capable of pharmaceutical comprising a methodfor reducing weight gain, controlling blood glucose level and controlabsorption of blood lipod.

[0035] The microorganisms that can be used for the pharmaceuticalcomposition of the present invention preferably fall within Acetobactergenus, Gluconobacter genus, Lactobacillus genus, and Acrobacteriumgenus, which are capable of living in the intestine and not harmful tohuman body, and are capable of converting oligosaccharides intopolysaccharides that cannot be absorbed into human body. Specifically,the following microorganisms can be used as microorganisms of thepharmaceutical composition of the present invention, such as,Acetobacter xylinum, A. hansenii, A. pasteurianus, A. aceti, Lactococcuscremoris, Lactobacillus helveticus, L. bulgaricus, L. sake, L. reutari,L. lactis, the subspecies of L. delbrueckii, L. delbrueckii subsp., anda variant form of L. helveticusglucose. Preferably, the microorganismscan be used as an active principle of the pharmaceutical composition ofthe present invention is Lactobacillus sp. BC-Y009 (KCTC0774BP) strainor Acetobacter sp. BC-Y058 (KCTC0773BP) strain.

[0036] The pharmaceutical composition of the present invention may beadministered in a form of tablet, capsule, suspension or emulsion, whichcomprises excipients, pharmaceutically acceptable vehicles and carrierswhich are selected depending on administration routes. Thepharmaceutical formulation of the present invention may furthercomprises supplemental active ingredients.

[0037] Lactose, dextrose, sucrose, sorbitol, mannitol, starch, acaciagum, calcium phosphate, alginic acid salt, treguhkense latex, gelatin,calcium silicate, finecrystalline cellulose, polyvinylpyrolidon,cellulose, water, syrup, methylcellulose, methylhydroxybenzoate andprophylhydroxybenzoate, talc, magnesium stearate or mineral oil may beused as carriers, exipients or diluents in the pharmaceuticalcomposition of the present invention.

[0038] In addition, the pharmaceutical composition of the presentinvention may further comprises lubricants, moisturizer, emulsifier,suspension stabilizer, preservative, sweetener and flavor. Thepharmaceutical composition of the present invention may be in a form ofan enteric coating formulation produced by various methods which havebeen publicly known, in order to deliver the active ingredients of thepharmaceutical composition, i.e., microorganisms, to the smallintestines without degradation by gastric juices in stomach.

[0039] Furthermore, microorganisms of the present invention may beadministered in a form of capsule prepared by conventional process. Forexample, standard vehicles and lyophilized microorganisms of the presentinvention are mixed together and prepared to pellets and then, thepellets are filled into hard gelatin capsules. In addition, themicroorganisms of the present invention and pharmaceutically allowablevehicles, for example, aqueous gum, cellulose, silicate or oil are mixedto produce a suspension or emulsion and then, this suspension oremulsion may be filled into soft gelatin capsule.

[0040] The pharmaceutical composition of the present invention may beprepared as an enterically coated tablets or capsules for oraladministration. The term “the enteric coating” of this applicationincludes all conventional pharmaceutically acceptable coating that hasresistance to gastric juice, however, in the small intestines, candisintegrate sufficiently for a rapid release of the microorganisms ofthe present invention.

[0041] The enteric coating of the present invention can be maintainedfor more than 2 hours in synthetic gastric juice, such as HCl solutionof pH 1 at the temperature of 36° C. to 38° C. and desirably, decomposeswithin 0.5 hours in synthetic intestinal juice, such as KH₂PO₄ buffersolution of pH 6.8.

[0042] The enteric coating of the present invention applies to eachtablet with the amount of about 16 to 30 mg, desirably 16 to 25 mg, moredesirably 16 to 20 mg. The thickness of enteric coating of the presentinvention is 5 to 100 μm, desirably 20 to 80 μm. The components of theenteric coating are selected appropriately from common polymericmaterials which have been publicly well known. The polymeric materialswhich may be employed for enteric coating of the present invention areenumerated and described in the flowing articles [The Theory andPractices of Industrial Pharmacy, 3rd Edition, 1986, pp. 365-373 by L.Lachman, Pharmazeutische Technologie, thieme, 1991, pp. 355-359 by H.Sucker, Hagers Handbuch der Pharmazeutischen Praxis, 4th Edition, Vol.7, pp. 739, 742, 766, and 778, (SpringerVerlag, 1971), and Remington'sPharmaceutical Sciences, 13th Edition, pp. 1689 and 1691 (Mack Publ.,Co., 1970)]. For example, cellulose ester derivative, cellulose etherand copolymer of acryl and methyl acrylate or maleic acid or phthalicacid derivative may be used in enteric coating of the present invention.

[0043] The preferred enteric coating of the present invention areprepared from polymers of cellulose acetate phthals or trimelitate andmethacrylic copolymer (for example, copolymer of more than 40% ofmethacrylic acid and methacrylic acid which contains hydroxyprophylmethylcellulose phthalate or derivatives from ester thereof).

[0044] Endragit L 100-55 manufactured by Rohm GmbH of Germany may beused as a raw material for the enteric coating of the present invention.

[0045] Cellulose acetate phthalate employed in the enteric coating ofthe present invention, has about 45 to 90 cP of viscosity, 17 to 26% ofacetyl contents and 30 to 40% of phthalate contents. The celluloseacetate trimelitate used in the enteric coating, has about 15 to 21 cSof viscosity, 17 to 26% of acetyl contents, and 25 to 35% of trimelitylcontents. The cellulose acetate trimelitate which is manufactured by theEastman Kodak Company may be used as a material for the enteric coatingof the present invention.

[0046] Hydroxyprophyl methylcellulose phthalate used in the entericcoating of the present invention has molecular weight of generally20,000 to 100,000 dalton, desirably 80,000 to 130,000 dalton and has 5to 10% of hydroxyprophyl contents, 18 to 24% of metoxy contents, and 21to 35% of phthalyl contents. Cellulose acetate phthalate manufactured bythe Eastman Kodak Company can be used as a material for the entericcoating of the present invention.

[0047] Hydroxyprophyl methylcellulose phthalate used in the entericcoating of the present invention is HP50 which is manufactured by theShin-Etsu Chemical Co. Ltd., Japan. The HP50 has 6 to 10% ofhydroxyprophyl contents, 20 to 24% of metoxy contents, 21 to 27% ofprophyl contents, and molecular weight is 84,000 dalton. Anothermaterial for enteric coating manufactured by the Shin-Etsu Chemical Co.Ltd., is HP55. HP55 can also be used as material for the enteric coatingof the present invention. The HP55 has 5 to 9% of hydroxyprophylcontents, 18 to 22% of metoxy contents, 27 to 35% of phthalate contents,and molecular weight is 78,000 dalton.

[0048] The enteric coating of the present invention is prepared by usingconventional methods of spraying the enteric coating solution to thecore. Solvents used in the process of the enteric coating are alcoholsuch as ethanol, ketone such as acetone, halogenated hydrocarbon such asdichloromethane, or the mixture thereof. Softeners such asDi-n-butylphthalate and triacetin are added to the enteric coatingsolution in the ratio of 1 part coating material to about 0.05 or toabout 0.3 part softner.

[0049] A spraying process is preferably performed continuously, and theamount of materials sprayed may be controlled depending on the conditionof the coating process. Spraying pressure may be regulated variouslyand, generally, desirable result can be obtained under the pressure ofaverage 1 to 1.5 bar.

[0050] “The effective amount” of this specification means the minimumamount of the microorganisms of the present invention, which can reducethe amount of oligosaccharide absorbed into the body through theintestines of mammalian animals. The amount of microorganismsadministered into a body with the pharmaceutical composition of thepresent invention may be adjusted depending on the administration methodand the administration subject.

[0051] The composition of the-present invention may be administered onceor more per day on the subject. The unit of administration amount meansthat it is separated physically and thus is suitable for the unitadministration for the human subjects and all other mammalian animals.Each unit contains a pharmaceutically acceptable carrier and the amountof the microorganisms of the present invention which are effective intherapy.

[0052] An oral administration unit of an adult patient containsmicroorganisms of the present invention in an amount, desirably, 0.1 gor more, and the composition of the present invention contains 0.1 to 10g per one time administration, desirably 0.5 to 5 g. The effectiveamount of microorganisms of the present invention is 0.1 g per 1 day.

[0053] However, the administration amount can vary depending on theweight and the severity of obesity of the patient, supplemental activeingredients included and microorganisms used therein. In addition, it ispossible to divide up the daily administration amount and to administercontinuously, if needed. Therefore, range of the administration amountdoes not limit the scope of the present invention in any way.

[0054] The “composition” of the present invention means not only asmedicinal products but also to serve as functional foods and healthcomplementary foods.

[0055] In case of taking the composition of the present inventionperiodically, microorganisms form colony within the intestines andinterrupt absorption of oligosaccharide in the body competitively. Also,non-digestable fibers produced by microorganisms make a healthycondition for other useful microorganisms within the intestines andstimulate the intestinal activity. Consequently, the composition of thepresent invention functions to treat and prevent obesity and diabetesmellitus.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The above objects and other advantages of the present inventionwill become more apparent by describing in detail a preferred embodimentthereof with reference to the attached drawings, in which:

[0057]FIG. 1 is the graph illustrating the absorption rate of glucose bythe microorganisms of the present invention.

[0058]FIG. 2 is the graph illustrating the change of blood glucose levelafter taking the microorganisms of the present invention.

[0059]FIG. 3 is the graph illustrating the change of energy metabolismefficiency of obese mouse that has taken the microorganism of thepresent invention.

[0060]FIG. 4 is the graph illustrating the change of energy metabolismefficiency of control mouse that has taken the microorganism of thepresent invention.

[0061]FIG. 5 is the graph illustrating the change of the body weight ofobese mouse induced by pharmacological prescription.

[0062]FIG. 6 is the graph illustrating the change of the metabolicefficiency of obese mouse induced by pharmacological prescription.

[0063]FIG. 7 is the phylogenetic analysis diagram of LactobacillusBC-Y009 based on 16 s rRNA nucleotide sequence of the present invention.

[0064]FIG. 8 is the phylogenetic analysis diagram of LactobacillusBC-Y058 based on 16 s rRNA nucleotide sequence of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] Hereinafter, the present invention will be described more indetail.

[0066] The microorganisms which can be used in the pharmaceuticalcomposition of the present invention for preventing and treating obesityand diabetes mellitus, or in a method therefore, should satisfy therequirements of 1) being capable of proliferating within the intestinallayers, 2) being capable of absorbing oligosaccharide rapidly and ofconverting them into non-digestable or hardly digestable high molecularweight materials, such as fibrous materials, and 3) being harmless tohuman body and animals. All microorganisms that can satisfy the aboverequirements can be used as active principles of the pharmaceuticalcomposition of the present invention and for use of the pharmaceuticalcomposition, and may be obtained from the numerous microorganismdepository institutions in the world.

[0067] Therefore, the microorganisms of the pharmaceutical compositionof the present invention are Acetobacter xylinum, Acetobacter BC-Y058,Acetobacter hansenii, Acetobacter pasteurianus, Acetobacter aceti,Leuconostoc sp., Bacillus sp., Lactobacillus BC-Y009, Lactobacillusbrevis, Lactobacillus helveticus, Lactobacillus bulgaricus,Lactobacillus casei, Lactobacillus kefir, Lactobacillus keriranofaciens,Lactobacillus bifidus, Lactobacillus sake, Lactobacillus reuteri,Lactobacillus lactis, Lactobacillus delbrueckii, Lactobacillushelveticusglucos var. jugurti., Lactococcus cremoris, Bifidobacteriumbifidium, Streptococcus thermophilus or Pediococcus sp. Bacteria, whichproduce polysaccharide. These microorganisms are described in thefollowing Articles:

[0068] Bart Degeest and Luc De Vuyst,

[0069] “Indication that the Nitrogen Source Influences Both Amount andSize of Exopolysaccharides Produced by Streptococcus thermophilus LY03and Modelling of the Bacterial Growth and Exopolysaccharide Productionin a Complex Medium”

[0070] (Appl. Envir. Microbiol. 1999, 65: 2863-2870);

[0071] Stacy A. Kimmel, Robert F. Roberts and Gregory R. Ziegler,

[0072] “Optimization of Exopolysaccharide Production by Lactobacillusdelbrueckii subsp. bulgaricus RR Grown in a Semidefined Medium”

[0073] (Appl. Envir. Microbiol. 1998, 64: 659-664.);

[0074] P. L. Pham, I. Dupont, D. Roy, G. Lapointe and J. Cerning,

[0075] “Production of Exopolysaccharide by Lactobacillus rhamnosus

[0076] R and Analysis of Its Enzymatic Degradation during ProlongedFermentation”

[0077] (Appl. Envir. Microbiol. 2000, 66: 2302-2310.);

[0078] Petronella J. Looijesteijn, Ingeborg C. Boels, MichielKleerebezem and Jeroen Hugenholtz,

[0079] “Regulation of Exopolysaccharide Production by Lactococcus lactissubsp. cremoris by the Glucose Source”

[0080] (Appl Envir. Microbiol. 1999, 65: 5003-5008);

[0081] G. H. Van Geel-Schutten, E. J. Faber, E. Smit, K. Bonting, M. R.Smith, B. Ten Brink, J. P. Kamerling, J. F. G. Vliegenthart and L.Dijkhuizen,

[0082] “Biochemical and Structural Characterization of the Glucan andFructan Exopolysaccharides Synthesized by the Lactobacillus reuteriWild-Type Strain and by Mutant Strains”

[0083] (Appl. Envir. Microbiol. 1999, 65: 3008-3014.);

[0084] G. J. Grobben, I. Chin-Joe, V. A. Kitzen, I. C. Boels, F. Boer,J. Sikkema, M. R. Smith and J. A. M. de Bont,

[0085] “Enhancement of Exopolysaccharide Production by Lactobacillusdelbrueckii subsp. bulgaricus NCFB 2772 with a Simplified DefinedMedium”

[0086] (Appl. Envir. Microbiol. 1998, 64: 1333-1337.);

[0087] Sandrine Petry, Sylviane Furlan, Marie-Jeanne Crepeau, JuttaCerning and Michel Desmazeaud,

[0088] “Factors Affecting Exocellular Polysaccharide Production byLactobacillus delbrueckii subsp. bulgaricus Grown in a ChemicallyDefined Medium”

[0089] (Appl. Envir. Microbiol. 2000, 66: 3427-3431.);

[0090] Richard van Kranenburg, Iris I. van Swam, Joey D. Marugg, MichielKleerebezem and Willem M. de Vos,

[0091] “Exopolysaccharide Biosynthesis in Lactococcus lactis NIZO B40:Functional Analysis of the Glycosyltransferase Genes Involved inSynthesis of the Polysaccharide Backbone”

[0092] (J. Bacteriol. 1999, 181: 338-340.);

[0093] Deborah Low, Jeffrey A. Ahlgren, Diane Horne, Donald J. McMahon,Craig J. Oberg and Jeffery R. Broadbent,

[0094] “Role of Streptococcus thermophilus MR-1C CapsularExopolysaccharide in Cheese Moisture Retention”

[0095] (Appl Envir. Microbiol. 1998, 64: 2147-2151.);

[0096] Richard van Kranenburg and Willem M. de Vos,

[0097] “Characterization of Multiple Regions Involved in Replication andMobilization of Plasmid pNZ4000 Coding for Exopolysaccharide Productionin Lactococcus lactis”

[0098] (J. Bacteriol. 1998, 180: 5285-5290.);

[0099] F Stingele, J R Neeser, and B Mollet,

[0100] “Identification and characterization of the eps(Exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6”

[0101] (J. Bacteriol. 1996, 178: 1680-1690.);

[0102] M Kojic, M Vujcic, A Banina, P Cocconcelli, J Cerning and LTopisirovic,

[0103] “Analysis of exopolysaccharide production by Lactobacillus caseiCG11, isolated from cheese”

[0104] (Appl. Envir. Microbiol. 1992, 58: 4086-4088.);

[0105] Christian Chervaux, S. Dusko Ehrlich and Emmanuelle Maguin,

[0106] “Physiological Study of Lactobacillus delbrueckii subsp.bulgaricus Strains in a Novel Chemically Defined Medium”

[0107] (Appl. Envir. Microbiol. 2000, 66: 5306-5311.);

[0108] J Lemoine, F Chirat, J M Wieruszeski, G Strecker, N Favre and J RNeeser,

[0109] “Structural characterization of the exocellular polysaccharidesproduced by Streptococcus thermophilus SFi39 and SFi12”

[0110] (Appl. Envir. Microbiol. 1997, 63: 3512-3518.);

[0111] Bart Degeest and Luc De Vuyst,

[0112] “Correlation of Activities of the Enzymes-Phosphoglucomutase,UDP-Galactose 4-Epimerase, and UDP-Glucose Pyrophosphorylase withExopolysaccharide Biosynthesis by Streptococcus thermophilus LY03”

[0113] (Appl. Envir. Microbiol. 2000, 66: 3519-3527.);

[0114] Petronella J. Looijesteijn, Ingeborg C. Boels, MichielKleerebezem and Jeroen Hugenholtz,

[0115] “Regulation of Exopolysaccharide Production by Lactococcus lactissubsp. cremoris by the Glucose Source”

[0116] (Appl. Envir. Microbiol. 1999, 65: 5003-5008.);

[0117] G. J. Grobben, I. Chin-Joe, V. A. Kitzen, I. C. Boels, F. Boer,J. Sikkema, M. R. Smith and J. A. M. de Bont,

[0118] “Enhancement of Exopolysaccharide Production by Lactobacillusdelbrueckii subsp. bulgaricus NCFB 2772 with a Simplified DefinedMedium”

[0119] (Appl. Envir. Microbiol. 1998, 64: 1333-1337.);

[0120] Richard van Kranenburg, Iris I. van Swam, Joey D. Marugg, MichielKleerebezem and Willem M. de Vos,

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[0170] “The Variable Relation of Oxygen Consumption to CelluloseSynthesis by Acetobacter xylinum”

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[0179] “Genetic organization of the cellulose in Acetobacter xylinium”

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[0191] “Regulation of exopolysaccharide production by Lactococcus lactissubsp. cremoris By the glucose source”

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[0236] “The role of amino acids in intensification of Bacillus subtilisexopolysaccharide biosynthesis in deep growth conditions”

[0237] (Mikrobiologiia. 1995, January-February; 64(1):44-50.); and

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[0240] (Boll. Chim. Farm. 1994, January; 133(1):3-18.),

[0241] which are hereby incorporated by reference in their entirety,including any drawings, as if fully set forth herein.

[0242] In addition, the present inventors have isolated and obtainednovel microorganisms which can be used as an active principle of thepharmaceutical composition of the present invention.

[0243] In order to isolate and obtain novel microorganisms which satisfythe requirements for an active principle of the pharmaceuticalcomposition of the present invention, the present inventors haveresearched as follows:

[0244] Samples of microorganisms collected from the glucose factorysewage and other locations were inoculated in MRS and BHS agar mediumscontaining cycloheximide, and then cultured. Colonies formed in agarmedium were then inoculated into MRS and BHS liquid medium and incubatedwithout shaking. Microorganisms that formed a matrix or a membrane shapeon top layers of the medium were selected. Formed membranes wereseparated and tested for whether or not the separated membranes weredecomposed by the intestinal digestive enzyme. The results determinedwhether non-digestable (or hardly digestable) high molecular-weightcompounds were produced or not. Among the microorganisms, BC-Y009 andBC-Y058 were selected for their high productivity of extracellularpolysaccharide (dietary fiber).

[0245] Upon observing the morphology of BC-Y009 and BC-Y058 andcomparing with 16 s rRNA's partial DNA sequences, it was confirmed thateach showed high percentage of homology sequence when compared withLactobacillus and Acetobacter. Based on the phenotype and 16 s rRNA DNAsequence analysis, it was ascertained that BC-Y009 is a novelmicroorganism which falls within the Lactobaccilus genus and BC-Y058 asa novel microorganism of Acetobacter genus.

[0246] Lactobacillus BC-Y009 and Acetobacter BC-Y058 of the presentinvention were administered into a mouse which was induced to haveobesity and diabetes mellitus. The blood glucose level of a subjectmouse had been decreased approximately 70% after administration.

[0247] According to these results, it was confirmed that microorganismsof the present invention has an effect in decreasing blood glucose leveland thus it is effective for treating and preventing against diabetesmellitus.

[0248] When microorganisms of the present invention, BC-Y009 and BC-Y058were administered into a mouse induced to have diabetes mellitus andobesity, the feed consumption rate increased 17 to 24% upon comparisonwith a control mouse. However, weight gain versus feed consumptionamount was decreased. The result thus indicates that the microorganismcomposition of the present invention allows for humans to consumewithout worrying about obesity or diabetes mellitus.

[0249] From the observation that a blood lipid level is also lower thanthat of control group in case of taking these microorganisms, themicroorganisms of the present invention is found to be capable ofcontrolling the occurrence of diabetes mellitus, obesity and circulatorydiseases, for example, arteriosclerosis or myocardial infarction.Additionally, in case of a normal mouse, mouse administered with thecomposition of the present invention consumed more feed, thus energyefficiency had been decreased in comparison with a control mouse.However, it was confirmed that there was no side effects led from theadministration upon observing that the change of lipid content wasnegligible.

[0250] Hereinafter, the present invention will be further explained withreference to the following examples. The examples are given only forillustration of the invention and are not intended to limit the scope ofthe present invention.

EXAMPLE 1

[0251] Selecting of Microorganism Which Produces ExtracellularPolysaccharide from Samples

[0252] In order to isolate microorganisms which produce dietary fibers,samples were collected from glucose factory sewage and other locations.10 g of the mixture thus collected were disrupted and suspended in 90 mlof physiological saline solution (0.85% NaCl). The said suspendedsamples were diluted to 10⁻², 10⁻⁴, and 10⁻⁶ in physiological salinesolution. These diluted samples then smeared on MRS agar mediumcontaining 1 mg of cycloheximide per 100 ml medium (1% Peptone, 1% beefextract, 0.5% yeast extract, 2% glucose, 0.1% Tween-80, 0.2% Citric AcidAmmonium, 0.5% Sodium Acetate, 0.01% MgSO₄, 0.005% MnSO₄, 0.2% SodiumPhosphate pH6.5) and on BSH agar medium (2% glucose, 0.5% Peptone, 0.5%yeast extract, 0.27% Na₂HPO₄, 0.115% Citric Acid pH 5.0)(Hestirin andSchramm, J. Gen. Microbiol., 11:123, 1954) and cultured in 30° C. for 72hours. Approximately 2,000 colonies were selected and were initiallyinoculated in 5 ml MRS liquid medium and BSH liquid medium at 30° C. for72 hours and cultured without shaking. The microorganism which form amembrane shape on upper layer of the liquid medium and the microorganismwhich form capsule-shaped extracellular polysaccharide and of whichmedium was transparent, were selected. These microorganisms wereinoculated again in 5 ml of MRS liquid medium and BSH liquid medium andstirred at 30° C. and the absorbance thereof was measured at 600 nm byspectrophotometer. Microorganisms were diluted with BSH liquid mediumuntil the absorbance thereof reached to 0.2. 10 ml of microorganism thusdiluted was inoculated into 100 ml of BSH liquid medium at 30° C. for 72hours and cultured without shaking.

[0253] In order to measure the amount of extracellular polysaccharide(dietary fibers) thus produced, each medium were centrifuged at 6,000rpm in 4° C. to obtain the precipitation of microorganisms. Cellmembrane were disrupted by alkali lysis in 0.1 N NaOH solution and leftalone in 800° C. for 30 minutes and centrifuged at 6,000 rpm in 4° C.and repeated multiple times, the above process in entirety.Extracellular polysaccharide entangled like white strings were isolatedand lyophilized to be measured the amount thereof. Microorganisms withhigh extracellular polysaccharide productivity were selected andextracellular polysaccharide productivity was compared with each other(Table 1). TABLE 1 Comparison of extracellular polysaccharideproductivity Amount of produced extracellular Selection Numberpolysaccharide(dryweight g/l BSH) BC-Y 009 3.8 BC-Y 002 4.2 BC-Y 015 3.2BC-Y 026 4.1 BC-Y 058 4.8 BC-Y 112 3.0 BC-Y 130 3.4 BC-Y 201 3.3

EXAMPLE 2

[0254] The Morphological Determination and Characteristics of theSelected BC-Y009 and BC-Y058

[0255] Microorganisms which show high polysaccharide productivityselected from the Example 1 were BC-Y009, BC-Y002, BC-Y015, BC-Y026,BC-Y058, BC-Y112, BC-Y130, and BC-Y201. Upon observing partial DNAsequences, BC-Y009, BC-Y002, BC-Y015 and BC-Y026 were microorganisms ofLactobacillus genus, and BC-Y058, BC-Y112, BC-Y130 and BC-Y201 weremicroorganisms of Acetobacter genus.

[0256] Among these bacteria, BC-Y009 and BC-Y058 which show highpolysaccharide productivity were inoculated in MRS and BSH liquidmediums at 30° C. for 72 hours and cultured in suspension. Culturedmediums were centrifuged at 6,000 rpm in 4° C. to obtain microorganismsand the nucleic acids thereof were isolated by means of using theCTAB/NaCl method. By using 16 s rRNA consensus primer, 16 s rRNA wasamplified by means of PCR method, and the sequence thus obtained, wasdetermined. BLAST analysis (NCBI, USA) on the sequence thus determined,was performed and its result showed high percentage of sequence homologywith sequence of Lactobacillus hilgardii, Acetobacter xylinum,Gluconobacter sp., numerous other Lactobacillus sp. and Acetobactersp.(Tables 2 and 3). TABLE 2 Comparison of 16S rRNA nucleotide sequenceof Lactobacillus sp. BC-Y 009 L. delbrueckii L. subsp. L. helveticus L.acidophillus hilgardii Lactobacillus sp. BC-Y009 ATCC9649 NCDO2712TATCC4356 NCDO264 ATCC13133 BC-Y009 — 145 136 146 3 4 L. delbrueckii88.93 — 76 73 142 143 sp. ATCC9649 L. helveticus 89.16 93.94 — 21 134134 NCDO2712T L. acidophillus 88.85 94.43 98.33 — 144 144 ATCC4356 L.hilgardii 99.77 89.07 89.26 88.93 — 1 NCDO264 Lactobacillus sp. 99.6988.97 89.21 88.90 99.92 — ATCC13133

[0257] Among 1,400 base pairs which are included in comparison, topright of table indicates number of base pairs which show difference,bottom left of table indicates % homology TABLE 3 Comparison of 16S rRNAnucleotide sequence of Acetobacter sp. BC-Y 058 BC-Y 058 A.diazotrificus A. liafaciens A. hansenii A. xylinum A. europaeus BC-Y 058— 37 34 10 13 14 A. diazotrificus 97.20 — 17 37 35 36 A. liafaciens97.42 98.71 — 34 32 33 A. hansenii 99.24 97.20 97.42 — 15 16 A. xylinum99.02 97.35 97.58 98.86 — 3 A. europaeus 98.94 97.27 97.50 98.79 99.77 —

[0258] Among 1,320 base pairs which are included in comparison, topright of table indicates number of base pairs which show difference,bottom left of table indicates % homology

[0259] BC-Y009 is a gram-positive bacteria and 0.5 to 3.0 μm in size. Itis a non-motile & short-rod shaped bacteria. It does not form spores andis facultative anaerobic. The growth temperature is between 20° C. to37° C. and pH level is 2.0 to 8.0 and optimal pH level is 4.0 to 7.0.The experimental results showed that this microorganism was condensed inmilk and was negative (non-reactive) to catalase and formed whitecolored colony in complex medium. It was precipitated in MRS liquidmedium and BSH liquid medium in form of white colored capsule. Theturbidity of the liquid medium was clear and the microorganism producedextracellular polysacchardie in clear medium and in case liquid mediumwas shaken, the extracellular polysacchride (dietary fiber) were brokeninto small particles.

[0260] BC-Y058 is a gram-negative bacteria, rod shaped bacteria and 0.6to 0.8 μm in size and exists as single or a pair. It is also anon-motile and does not form spores. Growth rate thereof is slow,therefore 5 to 7 days of incubation time is needed and colonies formedare small and hard. In liquid medium, clear cellulose pellicle isformed. Ethanol, acetic acid, or lactic acid can be used as substratesand showed positive response to catalase. This microorganism producesacid by using glucose and in Hoier medium, it can not grow.

[0261] Upon consideration of the result of analysis of phenotype and 16s rRNA DNA sequence, BC-Y009 was named as Lactobacillus sp. BC-Y009 andBC-Y058 as Acetobacter sp. BC-Y058. They were deposited in KCTC(KoreanCollection for Type Cultures) on May 30, 2000, and the deposit numberwere granted as KCTC BC-Y009, KCTC BC-Y058, respectively.

EXAMPLE 3

[0262] The Degree of Decomposition of Extracellular Polysacchride(Dietary Fiber) by Intestinal Digestive Enzymes

[0263] In order to determine whether or not dietary fiber produced bysaid microorganisms is decomposed by intestinal digestive enzyme, 1 g ofporcine pancreatin that shows the activity of 3×U.S. Pharmacopia(manufactured by Sigma) and comprises amylase, lipase, protease andnuclease, was suspended in buffer solution (pH7.5) of 1 g of drieddietary fiber. This suspension was incubated for 7 days at 40° C. andthe suspension was collected once a day and the glucose therein wasanalyzed quantitatively by using DNS (3,5-dinitrosalicylic acid). Theresult thereof showed that dietary fibers has never been decomposed atall.

[0264] Therefore, it was confirmed that the dietary fibers produced bythe microorganisms of the present invention do not decompose within theintestine.

EXAMPLE 4

[0265] Glucose Absorption Rate of Bacteria

[0266] Glucose absorption rates of Lactobacillus acidophilus (KCTC3140),L. hilgardii (KCTC3500) known as probiotics, and the said LactobacillusBC-Y009, Acetobacter BC-Y002, Acetobacter BC-Y058 and E. coli., weremeasured in the condition of the intestine. The results are representedin FIG. 1 and Table 4.

[0267] As illustrated in FIG. 1 and Table 4, the microorganisms of thepresent invention are superior to the other lactic acid bacteria interms of glucose absorption rate. TABLE 4 Glucose concentrationdecreased by the bacteria of unit O.D. per unit time. glucose initialconcentration glucose glucose decreased per con- concentration unit timeand initial centration after 1 unit O.D. O.D.600 nm (mM) hour (mM)(mM/hr/O.D.) E.coli 3.0 ± 0.1 110   85 ± 0.5  8.3 ± 0.44 BC-Y009 3.0 ±0.2 110   50 ± 0.3   20 ± 1.5 BC-Y002 3.0 ± 0.1 110   30 ± 0.7 26.6 ±1.1 BC-Y058 3.0 ± 0.2 110 38.6 ± 0.3 23.8 ± 0.1 KCTC3500 3.0 ± 0.2 11067.2 ± 0.3 14.2 ± 0.4 KCTC3140 3.0 ± 0.1 110 65.2 ± 0.4 14.4 ± 0.1

EXAMPLE 5

[0268] Concentration and Survival Rate of Microorganisms in theIntestine After Adminstering Microorganisms

[0269] Mouse C57BL/6J Lep^(ob) ob/ob genetically induced of obesity anddiabetes mellitus (hereinafter, “OB Mouse”), was starved for 18 hoursand fed the composition of the present invention (the number ofmicroorganism of the composition was 1.0×10¹³ CFU/g) containing 1% ofLactobacillus BC-Y009, Acetobacter BC-Y058 (w/w, drying weight) for 7days, and then the bacterial concentration in the duodenum, the jejunum,and the large intestine of these mice were analyzed. In addition, thebacterial concentration in the duodenum, the jejunum, and the largeintestine of the control OB mouse that had been fed the feed withoutcontaining the microorganisms of the present invention, was analyzed.

[0270] In order to measure the amount of Lactobacillus, the duodenum,the jejunum, and the large intestine of the mouse that had been fedLactobacillus feed and the control mice were cut out. Each surfaces ofthe organs were rinsed with physiological saline solution and thecontents were suspended in physiological saline solution.. Then,inoculated in MRS agar medium and incubated at 37° C. Three (3) dayslater, the amount of bacteria was measured by counting floc and bysubtracting the amount of Lactobacillus in the control group todetermine the change of the amount of bacteria (Table 5).

[0271] In order to confirm the existence of Acetobacter, the each organsof mouse were cut out, then rinsed the surfaces of the organs withphysiological saline solution. The contents were suspended inphysiological saline solution, then inoculated in BSH liquid medium andcultured at 37° C. for 3 days. By checking the pellicle appeared on toplayer of the liquid medium, the existence of fiber-producing Acetobacterwas confirmed (Table 6).

[0272] According to the results represented in Table 5 and Table 6, thesaid two kinds of microorganisms were both able to proliferate in theintestine. TABLE 5 The amount of Lactobacillus sp. in the duodenum, thejejunum, and the large intestine of mouse Existence of the region ofbacterial membrane intestine weight (g) number (CFU/g) formationDuodenum 0.18 ± 0.03 83 ± 20 no Jejunum 0.29 ± 0.05 1.2 × 10³ ± 50 nolarge intestine 0.36 ± 0.07 5.1 × 10³ ± 30 yes

[0273] TABLE 6 The amount of Acetobacter sp. in the duodenum, thejejunum, and the large intestine of mouse existence of the region ofintestine weight (g) membrane formation duodenum 0.20 ± 0.02 no jejunum0.28 ± 0.04 yes large intestine 0.35 ± 0.03 yes

EXAMPLE 6

[0274] The Change in Blood Glucose Level Upon Feeding of BC-Y009 andBC-Y058

[0275] 100 g of mouse feed purchased from SAMYANG Co. and 400 g ofKorean rice were mixed to make a composition in which carbohydratecontent was 60%, then 5 g of dried Lactobaccillus BC-Y009 or AcetobacterBC-Y058 were added thereto to prepare a lyophilized tablet. Mice werefed this tablet with water.

[0276] All mice tested in this Example were female and OB mice.Acetobacter feed group (OB-058), Lactobacillus feed group (OB-009), andthe control group (OB-con, which has no microorganism of the presentinvention in the feed) were bred separately. The breeding condition wasthat there was light every 12 hour intervals (9:00-21:00 lighted,21:00-9:00 no lighted) and maintained 20 to 24° C. and 40 to 60%humidity.

[0277] Additionally, enteric coating solution was sprayed on driedLactobacillus BC-Y009 or Acetobacter BC-Y058 to produce the compostionof the present invention which comprises enteric coated microorganisms.The weight of the enteric coating of material on the composition wasapproximately 16 to 30 mg or less per tablet. The materials for theenteric coating were selected from common high molecular weightmaterials, such as, cellulose acetate phthalate, trimelitate, copolymerof methacrylic acid (Methylacrylic acid 40% or more, especiallymethylacrylic acid including hydroxypropyl methylcellulose phthalate andits ester derivatives), or mixture thereof.

[0278] Methylacrylate used in the Example was Endragit L 100-55manufactured by Rohm GmbH (Germany), cellulose acetate phthalate withabout 45 to 90 cP of viscosity, 17 to 26% of acetyl content and 30 to40% of phthalate content, or cellulose acetate trimelitate manufacturedby the Eastman Kodak Company (approximately 15 to 20 cS of viscosity, 17to 26% of acetyl content and 25 to 35% of trimelityl content).

[0279] The enteric coating was produced by a conventional coatingprocess wherein the enteric coating solution was sprayed on a core.Ethanol and acetone mixture was used as solvent and a softening agentwas added to the coating solution in a ratio of 1 to approximately 0.005or 0.3.

[0280] The enteric coating composition of the present invention producedby means of the process was provided to the mice with water forunrestricted taking. The blood glucose level of the mouse which hastaken the enteric coating composition, was measured.

[0281] Before measuring the blood glucose level of each mouse group,each mouse was starved for 18 hours. Following 60 minutes afterstarvation, sufficient amounts of feed were provided and after a 60minute period, serum was collected from the retroorbital venous plexusby using anti-coagulating agent-free capillary tubes.

[0282] The blood glucose level was measured by absorbance at 505 nm,using the Trinder kit (Cat. 315-500, Sigma, USA) which employs enzymecoloring method. The statistical error of the results was indicated byaverage±standard deviation per experimental group, and statisticalsignificance of the average difference in each group was tested throughANOVA (p<0.02).

[0283] Data for blood glucose level are illustrated in FIG. 2. Asillustrated in the FIG. 2, the blood glucose level for OB-con group isapproximately 500 mg/dl, whereas OB-058 blood glucose level is low.Additionally, due to administration of Acetobacter BC-Y058 andLactobacillus BC-Y009, the blood glucose levels of each mouse had beendecreased to approximately 70% and 53% each (Table 7). TABLE 7 Thechange of blood glucose level after administration of AcetobacterBC-Y058 and Lactobacillus BC-Y009 OB-009 OB-058 OB-con Blood glucose 229± 16 141 ± 19 492 ± 60 level(mg/dl)

EXAMPLE 7

[0284] The Change of Weight and Amount of Diet Due to Taking BC-Y058 andBC-Y009 and in Metabolic Efficiency

[0285] Mice were classified as OB-058 group, OB-009 group, OB-con group,and Acetobacter BC-Y058 and Lactobacillus BC-Y009 were administered oneach group and the weight of each mouse was measured in weeklyintervals. Along with the measuring of changes in weight, the weight offeed consumed by the mice was also measured, therefore changes ofmetabolic efficiency of each group were investigated.

[0286] The difference of weight change was apparent in each specieswhose genetic characteristics were different, but the difference ofweight change, within the group having the same genetic characteristicswas negligible.

[0287] As indicated in Table 8, the weight change of OB mice within theperiod of 7 weeks, regardless of the administration of AcetobacterBC-Y058 or Lactobacillus BC-Y009, was approximately 47% increase ofweight. However, on the contrary, as indicated in Tables 9 and 10, feedconsumption percentage, depending on microorganism administration,increased 17 to 24% in OB mice group.

[0288] That is, the weight increase of the mice fed feed which comprisesthe microorganisms of the prevent invention was the same as that of themice fed that does not contain the microorganism of the preventinvention. The results indicate that because Acetobacter BC-Y058 andLactobacillus BC-Y009 suppress increase of blood glucose levels aftermeal, increase of feed consumption occurs as its compensation. In otherwords, with the same amount of feed, increase of weight can be decreasedby feeding the microorganism of the present invention without causing nofurther weight increase because of lower metabolic efficiency. Becauseof the conversion of glucose into dietary fiber by BC-Y058 and BC-Y009microorganism, metabolic efficiency has changed.

[0289] According to the formula represented below, the change of energyefficiency depending on feed consumption, was calculated and representedin Table 10.

energy metabolic efficiency=(weight gain(g)/amount of feeding(g))×1,000

[0290] As represented in Table 10, when microorganisms were administeredto OB mouse, the energy metabolic efficiency was from 75 to 85% (FIG. 3)compared to that of the control group which was not administered withthe microorganisms of the present invention (FIG. 4). TABLE 8 Change ofthe mouse weight (g) 1 2 3 4 5 6 7 week week week week week week weekOB- 21.5 ± 26.53 ± 31.52 ± 34.91 ± 37.6 ± 40.1 ± 41.4 ± 009  3.21 2.723.01  2.5  2.53  1.74  1.47 OB- 21.95 ± 26.75 ± 31.65 ± 35.8 ± 38.25 ±40.35 ± 41.25 ± 058  5.3 4.60 2.33  1.27 0.78 0.64 0.21 OB- 21.4 ± 26.3± 31.9 ± 35.8 ± 38.35 ± 40.1 ± 41.75 ± con  2.83  1.56 0.99  2.12 2.33 2.69 3.61

[0291] TABLE 9 Change of amount of feed consumption according to theadministration of Acetobacter BC-Y058, Lactobacillus BC-Y009 (g) 0-16days 16-21 days 21-34 days 34-41 days Total OB-009 146.3 32.4 110.7 38.6328 OB-058 157.4 34.3 115.3 41 348 OB-con 128.1 34.8 80.3 36.5 279.7

[0292] TABLE 10 Energy metabolic efficiency energy rate of Amount ofweight gain metaboliceffi- average weight feed (g) (g) ciency weight (g)increase OB-009 328 9.9 121 41.4 0.48 OB-058 348 19.3 111 41.25 0.47OB-con 279.7 20.35 146 41.75 0.49

EXAMPLE 8

[0293] Change of Weight and Diet Amount of Obesity Mouse Induced by GTGand Subsequent Change in Metabolic Efficiency

[0294] Before feeding Acetobacter BC-Y058 and Lactobacillus BC-Y009,each mouse was administered with 1 g/kg of goldthioglucose (Cat. A-0632,Sigma, USA) in order to induce obesity. And every 3 or 4 weeks, weightchange was measured and only obesity-induced mice were selected. Foraccuracy of the experiment, a mouse of which weight increase was toogreat or too little relatively, was excluded from the experiment.

[0295] The target was female C57BL/6J mice and breeding environment andconditions were the same as those in Example 6. The test subjects wereclassified into BC-Y058 group, KCTC3140 group, KCTC3500 group, andBC-Y009 group depending on microorganisms.

[0296] The weight changes of mice depending on microorganismsadministered with, are illustrated in FIG. 5 and it is confirmed thatwhen Acetobacter BC-Y058 and Lactobacillus BC-Y009 were administered,the weight increase rate has decreased.

[0297] Additionally, as represented in Table 11 and FIG. 6, in case thatKCTC3140 and KCTC3500 which consume glucose but do not produce dietaryfibers, were administered, the energy efficiency of obesity-induced micewas higher than that of the control group which was not administeredwith the microorganisms of the present invention. However, the mousegroup which was administered with BC-Y009 and BC-Y058 which producedietary fibers, showed relatively low energy efficiency, especially incase of BC-Y058. That is the energy efficiency decreased to 55% comparedwith that of the control group (Table 12). TABLE 11 Metabolismefficiency of obese mouse induced with drug administration(g) energymetabolic Weight gain(g) amount of feed(g) efficiency Carbohydrate 5.43103.7 52 KCTC3140 6.65 92.4 72 KCTC3500 5.67 102.2 55 BC-Y009 4.38 104.442 BC-Y058 2.98 102.7 29

EXAMPLE 9

[0298] Lipid Level Changes When BC-Y058 and BC-Y009 Were Administered

[0299] After administration of the microorganisms of the presentinvention, the change of blood lipid, especially cholesterol change, wasanalyzed and confirmed whether or not the microorganisms affected thecirculatory disease, such as, artheriosclerosis and myocardialinfarction besides diabetes mellitus and obesity.

[0300] Lipid analysis was performed by means of enzyme coloring methodas in Example 6, using TG-glycezyme-V (Young-Yeoun Chemical Co., Japan),HDL-zyme-V (Young-Yeoun Chemical Co., Japan), Cholestezyme-V(Young-Yeoun Co., Japan), LDL cholesterol (Cat. 61 532, BioMeriux,France), to measure the absorbance at 505 to 570 nm with standardsolution, and the amount of lipid in blood was calculated.

[0301] As represented in Table 12, lipid concentration before feedadministration did not show any differences in obese mouse. However,after Acetobacter BC-Y058 and Lactobacillus BC-Y009 were administered,as indicated in Table 12, the change of lipid concentration was apparentafter 7 weeks.

[0302] In case of obese mice that have taken the microorganism, thelipid level did not change in comparison with the data of early steps inthe present experiment and however, in case of control mouse which hadnot been administered with the microorganisms, overall lipid content inblood was increased. TABLE 12 Lipid amount in blood beforeadministration of feed(mg/dl) total cholesterol TG HDL-C LDL-C OB-009130.22 ± 4.11  98.1 ± 11.4  98.73 ± 9.7 4.18 ± 2.36 OB-058 129.37 ± 4.24101.6 ± 10.36 113.52 ± 15.47 3.35 ± 2.08 OB-con 127.57 ± 4.32 97.13 ±14.64  96.86 ± 7.61 6.62 ± 2.78

[0303] TABLE 13 Lipid amount in blood after administration offeed(mg/dl) Total cholesterol TG HDL-C LDL-C OB-009  167.04 ± 1.12100.76 ± 3.2 157.71 ± 2.4  4.2 ± 2.08 OB-058 *135.25 ± 2.47  98.5 ± 2.83  135 ± 1.41 3.36 ± 1.31 OB-con   *174 ± 1.41  110.5 ± 1.06 165.25 ±1.06 3.19 ± 0.36

[0304] The Industrial Applicability of the Present Invention

[0305] The microorganisms of the present invention are capable of livingwithin the intestine and converting monosaccharides and disaccharidesinto high molecular weight materials which cannot be absorbed and hardlydigestible in the intestine, thereby remarkably reducing the amount ofmonosaccharide to be absorbed. Therefore, the energy required formetabolic activity is provided from lipids and protein accumulated inthe body, thus effectively suppressesing obesity and diabetes mellitus.In addition, the microorganisms of the present invention produce dietaryfibers within the intestine and excreting harmful materials along withthese dietary fibers, to prevent appendicitis or large intestinalcancer, to suppress cholesterol absorption and to clean the intestine.

[0306] While the present invention has been particularly shown anddescribed with reference to particular examples thereof, it will beunderstood by those skilled in the art that various changes in form anddetails may be conceived therefrom without departing from the spirit andscope of the present invention as defined by the appended claims.

[0307] This application claims priority from the Korean PatentApplication Nos. 10-2000-0026379 (filed May 17, 2000) and10-2000-0049805 (filed Aug. 26, 2000), the contents of which are herebyincorporated by reference in their entirety, including thespecification, drawings and claims.

What is claimed is:
 1. Lactobacillus sp. BC-Y009 (KCTC-0774BP). 2.Acetobacter sp. BC-Y058 (KCTC-0773BP).
 3. A pharmaceutical compositioncomprising at least one microorganism selected from the group consistingof Acetobacter sp., Leuconostoc sp., Bacillus sp., Lactobacillus sp.,Streptococcus sp., Bifidobacterium sp., Lactococcus sp. and Pediococcussp. bacteria in an amount effective to prevent or treat obesity and apharmaceutically acceptable carrier, wherein the microorganism iscapable of producing polysaccharide.
 4. The pharmaceutical compositionaccording to claim 3, wherein said microorganism is selected from thegroup consisting of Acetobactor sp., Lactobacillus sp. and Lactococcussp. bacteria.
 5. The pharmaceutical composition according to claim 3,wherein said microorganism is selected from the group consisting ofAcetobacter xylinum, Acetobacter BC-Y058, Acetobacter hansenii,Acetobacter pasteurianus, Acetobacter aceti, Leuconostoc sp., Bacillussp., Lactobacillus BC-Y009, Lactobacillus brevis, Lactobacillushelveticus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacilluskefir, Lactobacillus keriranofaciens, Lactobacillus bifidus,Lactobacillus sake, Lactobacillus reuteri, Lactobacillus lactis,Lactobacillus delbrueckii, Lactobacillus helveticusglucos var. jugurti.,Lactococcus cremoris, Bifidobacterium bifidium, Streptococcusthermophilus and Pediococcus sp.
 6. The pharmaceutical compositionaccording to claim 3, wherein said microorganism is selected from thegroup consisting of Acetobacter BC-Y058 and Lactobacillus BC-Y009. 7.The pharmaceutical composition according to claim 3, which is aformulation suitable for oral administration.
 8. The pharmaceuticalcomposition according to claim 3, which is a formulation coated withenteric coating materials.
 9. The pharmaceutical composition accordingto claim 7, which is a formulation coated with enteric coatingmaterials.
 10. A pharmaceutical composition comprising at least onemicroorganism selected from the group consisting of Acetobacter sp.,Leuconostoc sp., Bacillus sp., Lactobacillus sp., Streptococcus sp.,Bifidobacterium sp., Lactococcus sp. and Pediococcus sp. bacteria in anamount effective to prevent or treat diabetes mellitus and apharmaceutically acceptable carrier, wherein the microorganism iscapable of producing polysaccharide.
 11. The pharmaceutical compositionaccording to claim 10, wherein said microorganism is selected from thegroup consisting of Acetobactor sp., Lactobacillus sp. and Lactococcussp. bacteria.
 12. The pharmaceutical composition according to claim 10,wherein said microorganism is selected from the group consisting ofAcetobacter xylinum, Acetobacter BC-Y058, Acetobacter hansenii,Acetobacter pasteurianus, Acetobacter aceti, Leuconostoc sp., Bacillussp., Lactobacillus BC-Y009, Lactobacillus brevis, Lactobacillushelveticus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacilluskefir, Lactobacillus keriranofaciens, Lactobacillus bifidus,Lactobacillus sake, Lactobacillus reuteri, Lactobacillus lactis,Lactobacillus delbrueckii, Lactobacillus helveticusglucos var. jugurti.,Lactococcus cremoris, Bifidobacterium bifidium, Streptococcusthermophilus and Pediococcus sp.
 13. The pharmaceutical compositionaccording to claim 10, wherein said microorganism is selected from thegroup consisting of Acetobacter BC-Y058 and Lactobacillus BC-Y009. 14.The pharmaceutical composition according to claim 10, which is aformulation suitable for oral administration.
 15. The pharmaceuticalcomposition according to claim 10, which is a formulation coated withenteric coating materials.
 16. A method for preventing or treatingobesity, comprising administering to a subject in need thereof apharmacuetical composition comprising at least one microorganismselected from the group consisting of Acetobacter sp., Leuconostoc sp.,Bacillus sp., Lactobacillus sp., Streptococcus sp., Bifidobacterium sp.,Lactococcus sp. and Pediococcus sp. bacteria in an amount effective toprevent or treat obesity and a pharmaceutically acceptable carrier,wherein the microorganism is capable of producing polysaccharide. 17.The method according to claim 16, wherein the pharmaceutical compositioncomprises at least one microorganism selected from the group consistingof Acetobactor sp., Lactobacillus sp. and Lactococcus sp. bacteria. 18.The method according to claim 16, wherein the pharmaceutical compositioncomprises at least one microorganism selected from the group consistingof Acetobacter xylinum, Acetobacter BC-Y058, Acetobacter hansenii,Acetobacter pasteurianus, Acetobacter aceti, Leuconostoc sp., Bacillussp., Lactobacillus BC-Y009, Lactobacillus brevis, Lactobacillushelveticus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacilluskefir, Lactobacillus keriranofaciens, Lactobacillus bifidus,Lactobacillus sake, Lactobacillus reuteri, Lactobacillus lactis,Lactobacillus delbrueckii, Lactobacillus helveticusglucos var. jugurti.,Lactococcus cremoris, Bifidobacterium bifidium, Streptococcusthermophilus and Pediococcus sp.
 19. The method according to claim 16,wherein the pharmaceutical composition comprises at least onemicroorganism selected from the group consisting of Acetobacter BC-Y058and Lactobacillus BC-Y009.
 20. The method according to claim 16, whereinthe pharmaceutical composition is a formulation suitable for oraladministration.
 21. The method according to claim 16, wherein thepharmaceutical composition is a formulation coated with enteric coatingmaterials.
 22. The method according to claim 20, wherein thepharmaceutical composition is a formulation coated with enteric coatingmaterials.
 23. A method for preventing or treating diabetes mellitus,comprising administering to a subject in need thereof a pharmacueticalcomposition comprising at least one microorganism selected from thegroup consisting of Acetobacter sp., Leuconostoc sp., Bacillus sp.,Lactobacillus sp., Streptococcus sp., Bifidobacterium sp., Lactococcussp. and Pediococcus sp. bacteria in an amount effective to prevent ortreat diabetes mellitus and a pharmaceutically acceptable carrier,wherein the microorganism is capable of producing polysaccharide. 24.The method according to claim 23, wherein the pharmaceutical compositioncomprises at least one microorganism selected from the group consistingof Acetobactor sp., Lactobacillus sp. and Lactococcus sp. bacteria. 25.The method according to claim 23, wherein the pharmaceutical compositioncomprises at least one microorganism selected from the group consistingof Acetobacter xylinum, Acetobacter BC-Y058, Acetobacter hansenii,Acetobacter pasteurianus, Acetobacter aceti, Leuconostoc sp., Bacillussp., Lactobacillus BC-Y009, Lactobacillus brevis, Lactobacillushelveticus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacilluskefir, Lactobacillus keriranofaciens, Lactobacillus bifidus,Lactobacillus sake, Lactobacillus reuteri, Lactobacillus lactis,Lactobacillus delbrueckii, Lactobacillus helveticusglucos var. jugurti.,Lactococcus cremoris, Bifidobacterium bifidium, Streptococcusthermophilus and Pediococcus sp.
 26. The method according to claim 23,wherein the pharmaceutical composition comprises at least onemicroorganism selected from the group consisting of Acetobacter BC-Y058and Lactobacillus BC-Y009.
 27. The method according to claim 23, whereinthe pharmaceutical composition is a formulation suitable for oraladministration.
 28. The method according to claim 23, wherein thepharmaceutical composition is a formulation coated with enteric coatingmaterials.
 29. A method for controlling or preventing weight gain,comprising administering to a subject in need thereof a pharmaceuticalcomposition comprising at least one microorganism selected from thegroup consisting of Acetobacter sp., Leuconostoc sp., Bacillus sp.,Lactobacillus sp., Streptococcus sp., Bifidobacterium sp., Lactococcussp. and Pediococcus sp. bacteria in an effective amount and apharmaceutically acceptable carrier, wherein the microorganism iscapable of producing polysaccharide.
 30. The method according to claim29, wherein the said microorganism is selected from the group consistingof Acetobacter BC-Y058 and Lactobacillus BC-Y009.
 31. The methodaccording to claim 29, wherein the pharmaceutical composition issuitable for oral administration.
 32. The method according to claim 29,wherein the pharmaceutical composition is coated with enteric coatingmaterials.
 33. A method for reducing weight gain, comprisingadministering to a subject in need thereof a pharmaceutical compositioncomprising at least one microorganism selected from the group consistingof Acetobacter sp., Leuconostoc sp., Bacillus sp., Lactobacillus sp.,Streptococcus sp., Bifidobacterium sp., Lactococcus sp. and Pediococcussp. bacteria in an effective amount and a pharmaceutically acceptablecarrier, wherein the microorganism is capable of producingpolysaccharide.
 34. The method according to claim 33, wherein the saidmicroorganism is selected from the group consisting of AcetobacterBC-Y058 and Lactobacillus BC-Y009.
 35. The method according to claim 33,wherein the pharmaceutical composition is suitable for oraladministration.
 36. The method according to claim 33, wherein thepharmaceutical composition is coated with enteric coating materials. 37.A method for controlling blood glucose level, comprising administeringto a subject in need thereof a pharmaceutical composition comprising atleast one microorganism selected from the group consisting ofAcetobacter sp., Leuconostoc sp., Bacillus sp., Lactobacillus sp.,Streptococcus sp., Bifidobacterium sp., Lactococcus sp. and Pediococcussp. bacteria in an effective amount and a pharmaceutically acceptablecarrier, wherein the microorganism is capable of producingpolysaccharide.
 38. The method according to claim 37, wherein the saidmicroorganism is selected from the group consisting of AcetobacterBC-Y058 and Lactobacillus BC-Y009.
 39. The method according to claim 37,wherein the pharmaceutical composition is suitable for oraladministration.
 40. The method according to claim 37, wherein thepharmaceutical composition is coated with enteric coating materials. 41.The method according to claim 37, wherein a normal blood glucose levelis not affected.
 42. A method for controlling absorption of blood lipid,comprising comprising administering to a subject in need thereof apharmaceutical composition comprising at least one microorganismselected from the group consisting of Acetobacter sp., Leuconostoc sp.,Bacillus sp., Lactobacillus sp., Streptococcus sp., Bifidobacterium sp.,Lactococcus sp. and Pediococcus sp. bacteria in an effective amount anda pharmaceutically acceptable carrier, wherein the microorganism iscapable of producing polysaccharide.
 43. The method according to claim42, wherein the said microorganism is selected from the group consistingof Acetobacter BC-Y058 and Lactobacillus BC-Y009.
 44. The methodaccording to claim 42, wherein the pharmaceutical composition issuitable for oral administration.
 45. The method according to claim 42,wherein the pharmaceutical composition is coated with enteric coatingmaterials.